Genome-Wide RNAi Screening Identifies Novel Pathways/Genes Involved in Oxidative Stress and Repurposable Drugs to Preserve Cystic Fibrosis Airway Epithelial Cell Integrity.

Javier Checa,Josep M Aran,Jose Luis Mosquera,Maria Maqueda,Itziar Martínez-González

doi:10.3390/antiox10121936

Abstract

Recurrent infection-inflammation cycles in cystic fibrosis (CF) patients generate a highly oxidative environment, leading to progressive destruction of the airway epithelia. The identification of novel modifier genes involved in oxidative stress susceptibility in the CF airways might contribute to devise new therapeutic approaches. We performed an unbiased genome-wide RNAi screen using a randomized siRNA library to identify oxidative stress modulators in CF airway epithelial cells. We monitored changes in cell viability after a lethal dose of hydrogen peroxide. Local similarity and protein-protein interaction network analyses uncovered siRNA target genes/pathways involved in oxidative stress. Further mining against public drug databases allowed identifying and validating commercially available drugs conferring oxidative stress resistance. Accordingly, a catalog of 167 siRNAs able to confer oxidative stress resistance in CF submucosal gland cells targeted 444 host genes and multiple circuitries involved in oxidative stress. The most significant processes were related to alternative splicing and cell communication, motility, and remodeling (impacting cilia structure/function, and cell guidance complexes). Other relevant pathways included DNA repair and PI3K/AKT/mTOR signaling. The mTOR inhibitor everolimus, the α1-adrenergic receptor antagonist doxazosin, and the Syk inhibitor fostamatinib significantly increased the viability of CF submucosal gland cells under strong oxidative stress pressure. Thus, novel therapeutic strategies to preserve airway cell integrity from the harsh oxidative milieu of CF airways could stem from a deep understanding of the complex consequences of oxidative stress at the molecular level, followed by a rational repurposing of existing “protective” drugs. This approach could also prove useful to other respiratory pathologies.

Highlights

IntroductionRespiratory dysfunction and failure have devastating consequences in cystic fibrosis (CF), the most frequent hereditary autosomal disease in the Caucasian population
Both flow cytometry and fluorescence microscopy confirmed that symmetric transcription from convergent polymerase III (Pol III) promoters induced efficient, dose-dependent
The self-perpetuating infection-inflammation cycle occurring in the lungs of cystic fibrosis (CF) patients constitutes a chronic challenge to the integrity of airway epithelial cells, oxidative stress being a key element contributing to persistent cellular damage and preventing proper airway remodeling [2,48]

Summary

Introduction

Respiratory dysfunction and failure have devastating consequences in cystic fibrosis (CF), the most frequent hereditary autosomal disease in the Caucasian population. CF is caused by mutations in the CFTR gene leading to absence or abnormal function of the CFTR anion channel in the surface of epithelial cells, its pathophysiology remains incompletely understood. Failure of chloride and bicarbonate secretion and concomitant sodium hyperabsorption at the airway apical surface induces dehydration of the superficial fluid layer and impairs mucociliary clearance [1,2,3].

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